Fermentation route for the production of levulinic acid, levulinate esters and valerolactone and derivatives thereof

ABSTRACT

The invention provides processes for the conversion of pyruvate obtained from sugars or other carbon sources, to valuable C5 materials such as levulinic acid, levulinate esters, valerolactone, and derivatives thereof.

PRIORITY

This application claims priority to U.S. Provisional Application No.61/378,199, filed Aug. 30, 2010, which is hereby incorporated byreference.

BACKGROUND

Levulinic acid, or 4-oxopentanoic acid, is an organic compound with theformula CH₃C(O)CH₂CH₂CO₂H. It is a keto acid. Levulinic acid istypically prepared chemically, for example, by heating sucrose withconcentrated hydrochloric acid. The process proceeds via theintermediacy of glucose, which is isomerized to fructose and thenhydroxymethylfurfural.

Levulinic acid is a potential precursor to nylon-like polymers,synthetic rubbers, and plastics. Levulinic acid is a versatile syntheticintermediate, e.g., in the synthesis of pharmaceuticals, and is aprecursor in the industrial production of other chemical commoditiessuch as methyltetrahydrofuran, valerolactone, and ethyl levulinate.

SUMMARY OF THE INVENTION

In certain aspects and embodiments, the invention provides a chemicalpathway for the conversion of pyruvate obtained from sugars or othercarbon sources, to valuable C5 materials such as levulinic acid.Exemplary C5 compounds are shown in FIGS. 1 and 2. When used with sugarsas a carbon source, the key to the pathway is to convert C6 sugars (suchas, but not limited, to glucose, fructose, galactose) and/or C5 sugars(such as, but not limited to, xylose, arabinose) into pyruvate, andsubsequently convert pyruvate into one or several valuable C5 compoundsthrough chemical or biochemical aldol addition, oxidation, reduction,dehydration and cyclization reactions. When used with another carbonsource such as, but not limited to, fatty acids and glycerol, the carbonsource is first converted into pyruvate, and subsequently converted toone or several valuable C5 compounds, which include linear C5 keto acidsor esters or cyclized derivatives thereof of the following generalformula: C₅C₄(X)C₃C₂(Y)C₁(═O)(Z), where X is either a hydroxyl or ketoneoxygen, Y is either a hydrogen, a hydroxyl or ketone oxygen, the bondbetween the C3 and C2 carbons is either single of double (e.g. saturatedor unsaturated) and Z is an alkoxy, sulfide or phenoxy group as to makeeither an ester, thioester or carboxylic acid functional group. In someembodiments, the C5 compound is C₅C₄(O₁)C₃C₂(Y)C₁(═O)(O₁), wherein theindice “O₁” denotes the same oxygen atom such that there is a cyclicester, or lactone formed, and Y is either a hydrogen or a hydroxyl orketone oxygen. All other atomic valences, or bonds, arc assumed to behydrogen atoms unless otherwise denoted above.

In one aspect, the invention provides a method for making a compoundthat is a C5 keto acid or ester, or a C5 hydroxy acid or ester, orcyclic derivative thereof. The method comprises converting pyruvate to aC5 intermediate by aldol addition, and converting the C5 intermediate tosaid compound through chemical or enzymatic steps or a combinationthereof. In certain embodiments, the C5 compound has the general formulaC₅C₄(X)C₃C₂(Y)C₁(═O)(Z) or C₅C₄(O₁)C₃C₂(Y)C₁(═O)(O₁) as described above.In certain embodiments, the compound is prepared from 5-carbon and/or6-carbon sugars or feedstock suitable as carbon source for a microbialhost. In these embodiments, the method comprises formation of pyruvatefrom the sugar or feedstock (e.g., by the microbial host), and aldoladdition of acetylaldehyde to the pyruvate (e.g., in the microbial hostor in a cell-free system), to thereby prepare a 5-carbon keto acid as anintermediate for the preparation of the desired C5 compound.Acetylaldehyde for aldol addition may be prepared by decarboxylation ofpyruvate in the microbial host. The aldol addition product may befurther subjected to one or more reduction, oxidation, dehydration,group transfer, hydrolysis and/or lactonization reactions (e.g., eachindependently in the microbial host or cell free system) to prepare thedesired C5 product.

Such products may be used as building blocks to prepare commerciallyvaluable chemicals and fuels. For example, lactones such as2-oxo-valerolactone (compound L7 in FIG. 2), 2-hydroxy-valerolactone(compound L6 in FIG. 2), angelica lactones (compound L2, L3 and L10 inFIG. 2) and 4-valerolactone (γ-valerolactone, compound L1 in FIG. 2) canbe used as solvents. Angelica lactone and 4-valerolactone can also beconverted chemically to methylene methyl butyrolactone (MeMBL) (see forexample WO/2006/015023, WO/2006/015024 for methods to catalyze thisconversion). Methylene methyl butyrolactonc can be used as a monomer orcopolymer to increase the thermal tolerance of polymethylacrylate (PMMA)polymers used widely in electronics and automotive applications, or tomanufacture polymers altogether (such as Poly(MeMBL), see for instanceWO/2005/028529). In addition, 4-valerolactone can be converted usingchemical catalysis to valeric acid and further valerate esters, as wellas isomeric butenes, butadiene and other alkenes, including alkenes ofeight carbons or more, as reviewed in Bozell J., Connecting Biomass andpetroleum Processing with a chemical bridge, Science 329:522-523 (2010).Levulinic Acid (compound P1 in FIG. 1) can be converted to 1,4pentanediol and diphenolic acid, both of which can be used tomanufacture polymers. δ-amminolevulinic acid (a derivative fromLevulinic Acid) is a herbicide with an estimate market in excess of 300pounds per year. Further still, Levulinic Acid can be converted topyrrolidones (WO/2004/085048), pyrrolidinone (WO/2010/065833,WO/2004/085390, WO/2004/085349, WO/2004/084633), angelica lactone(WO/2005/097723), 4-valerolactone and 2-methyl-THF, which are endproducts or can be further transformed into other compounds with variousutilities such as anionic liquids (WO/2010/065833), biofuels and fueladditives. Levulinic Acid can further be employed as a material forbatteries (e.g. JP09190820), inks (U.S. Pat. No. 5,769,929), coatings(JP06280041), anti-corrosion coatings (EP496555) Levulinic esters (orlevulinate esters, compounds P9 in FIG. 1) are polymer building blocksby themselves and after transformation to ketals (US 2008/0242721) andcan also be used as fuel additives (as described in U.S. Pat. No.7,153,996, which is hereby incorporated by reference in its entirety).In addition, levulinic esters can be used in personal care products(e.g. Japanese patent JP 05320023), surfactants and lubricants(EP882745), absorbents (see WO/1998/9843684). All references cited inthis paragraph are hereby incorporated by reference.

Both levulinic acid, levulinic esters, and some of the lactones listedin FIG. 2 can also be used in the manufacture of pharmaceutically activeingredients, and pharmaceutical applications, some of which being listedin Bozell J., Production of levulinic acid and use as a platformchemical for derived products, Resources, Conservation and Recycling28:227-239 (2000). For instance, WO/1995/022524 reports the use oflevulinate methyl ester for the synthesis of novel indole derivativesused as anti-cancer agents. Levulinic acid and 4-hydroxy-pentanoic acidcan also by used a chiral reagent, with a wide array of potentialapplications (see for example Meyers et al., Stereoselective alkylationsin rigid systems. Effect of remote substituents on p-facial additions tolactam enolates. Stereoelectronic and steric effects, J. Am. Chem. Soc.120:7429-7438 (1998). Pharmaceutical applications of the C5 produced bythe invention may include the use of butyro- and valero-lactonederivatives as antibiotic and anti-biofilms agents through thereinterference with the quorum sensing molecular mechanism in bacteria(see for instance EP 1716131 and WO/2006/117113). Additional uses mayderive from the biologically active proto-anemonin (compound L4 in FIG.2). Finally, Levulinic Acid and esters have been used for food, flavorand fragrances (EP1533364) as well as additives in numerous consumerproducts. For example, Levulinic Acid is used as an additive incigarettes (WO/2010/051076). All references in this paragraph are herebyincorporated by reference.

In certain embodiments of the invention, the method comprises convertingpyruvate into 4-valerolactone. In another embodiment, the methodcomprises converting pyruvate into levulinic acid. In anotherembodiment, the method comprises converting pyruvate into levulinicesters (levulinates) such as, but not limited to, ethyl levulinate andpropyl levulinate. In another alternative embodiment of the invention,the method comprises converting pyruvate into angelica lactone, alpha-and alpha′-angelica lactones. In still other embodiments, the methodcomprises converting pyruvate into 2,4-dihydroxy-pentanoic acid or itscyclized form, 2-hydroxy-4-valerolactone. In yet another embodiment, themethod comprises converting pyruvate into 2-oxo-4-hydroxy-pentanoic acidor its cyclized form, 2-oxo-4-valerolactone.

In some embodiments of the invention, the method comprises multipleenzymatic steps integrated into a single metabolic pathway in aeukaryotic, prokaryotic or archaea fermentation host, including but notlimited to Saccharamyces sp., Pichia sp., Pseudomonas sp., Bacillus sp.,Chrysosporium sp., and Escherichia coli. In these and other embodiments,the method involves one or more enzymatic steps carried out in acell-free system, or chemical catalysis steps, or a combination thereof,the various intermediates in the pathway being optionally separatedand/or purified from the fermentation broth as necessary to complete theprocess.

An advantage of certain embodiments of the invention is that it buildson top of central metabolism. For instance, both C5 and C6 metabolism ineukaryotes, prokaryotes and archea can employ glycolysis to producepyruvate. Pyruvate is one of the most important intermediates of centralmetabolism, and in addition to glycolysis can be obtained from lipidmetabolism as well as amino-acid metabolism. The method of the inventiontakes pyruvate, and converts two molecules of pyruvate into one C5molecule such as levulinic acid and 4-valerolactone. In the case of C6sugars the carbon yield can be up to 80%. In the case of C5 sugars thecarbon yield can be theoretically up to 100%. If the method employs amicrobial strain capable of simultaneously fermenting C5 and C6 such as,but not limited to, engineered Saccharomyces Cerevisae and PichiaStipitis, it allows the direct fermentation of sugars to levulinic acid,4-valerolactone or any of the C5 compounds depicted in FIG. 1 and FIG.2. This high achievable yield presents a decisive industrial advantagewhen compared to alternative thermochemical methods of obtaininglevulinic acid or gamma-valerolactone which typically produce molaryields of 40% or less.

In one embodiment of the invention, the method converts a stream ofsugars into one or several of the C5 compounds listed in FIGS. 1 and 2.In another embodiment, starch is used as feedstock for the process. Inanother embodiment, the method converts lignocellulosic feedstock(including, but not limited to, corn stover, wood chips, municipalwaste, Pulp and Paper mill sludge) into at least one of the C5 compoundslisted in FIGS. 1 and 2.

In one embodiment of the invention, the method converts C6 sugars intoone or several of the C5 compounds listed in FIGS. 1 and 2, preferablyin fermentation strains highly efficient at uptake and fermentation ofC6 sugars, such as, but not limited to, Saccharomyces cerevisiae,Cargill's CB1 strain (as described in WO/2007/106524), Pseudomonas,Chrysosporium and Escherichia coli (E. coli). In another embodiment ofthe invention, the method converts C5 sugars to one or several of the C5compounds listed in FIGS. 1 and 2, preferably in fermentation strainshighly efficient at uptake and fermentation of C5 sugars, such as, butnot limited to, engineered Saccharomyces Cerevisiae and Pichia stipitis.In an embodiment of the invention, the method simultaneously converts C5and C6 sugars to the C5 compound, preferably in fermentation strainshighly efficient at uptake and fermentation of both C5 and C6 sugars(e.g., Saccharomyces cerevisiae). In another embodiment of theinvention, the fermentation strains show high level of tolerance tobiomass hydrolysate inhibitors such as, but not limited to, furans andto low pH or high organic acid titer media.

In certain embodiments, the feedstock comprises one or more C6 sugarsselected from allose, altrose, glucose, mannose, gulose, idose, talose,galactose, fructose, psicose, sorbose, and tagatose. In these or otherembodiments, the feedstock comprises one or more C5 sugars selected fromxylose, arabinose, ribose, lyxose, xylulose, and ribulose.

When the method of the invention is used to convert C5 and C6 sugars to4-hydroxy-pentanoic acid or 4-valerolactone, the pathway is designed tobe redox (reduction-oxidation) balanced: two reducing equivalents(formation of NAD(P)H) are produced during glycolysis to yield pyruvate(one glucose molecule to two pyruvate molecules) and two reducingequivalents are consumed (formation of NAD(P)) by the downstream processfrom pyruvate to 4-hydroxy-pentanoic acid or 4-valerolactone(γ-valerolactone). The fact that this pathway is redox balanced for theproduction of these two molecules will result in optimized conversion inthe case of fermentation process, and reduce or remove the need tofurther engineer the fermentation host to counter inbalance. For thefermentation of sugars directly to all the other compounds or buildingblocks (FIG. 1), the fermentation host will rely on separate sidereactions to balance the pathway or an external source of redoxequivalent suitable to balance the pathway.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the molecular formulae for levulinic acid (compound P1 inFIG. 1), as well as valuable derivatives that can be produced atdifferent steps in the pathway and in various embodiments of theprocesses described herein (compounds P1 to P16 in FIG. 1).

FIG. 2 shows the molecular formulae for various C5 lactones that can beproduced at different steps in the pathway according to variousembodiments of the method (compounds L1 to L10).

FIG. 3 provides a general view of the biochemical processes convertingpyruvate to any of the C5 compounds of FIG. 1 or FIG. 2, with certainsteps highlighted. Some possible chemical intermediates and subroutesare not depicted here. See FIG. 5 for a more exhaustive depiction of thedifferent pathway possibilities.

FIG. 4 provides a general view of the biochemical pathway and processesaccording to certain embodiments of the invention, where the order ofthe oxidation/reduction steps (corresponding to steps 3 and 4 in FIG. 3)is inverted. As in FIG. 3, some possible chemical intermediates andsub-routes are not depicted here. See FIG. 6 for a more exhaustivedepiction of the different pathway possibilities different pathwaypossibilities.

FIG. 5 provides general view of the biochemical pathway/processes ofFIG. 3 where step 4 and step 5 are collapsed into one step using anoxidative dehydratase.

FIG. 6 provides a detailed view of the biochemical pathway/processesconverting pyruvate to any of the C5 compounds or building blocks ofFIG. 1 or 2. In addition to the chemical steps depicted in FIGS. 3, 4,and 5, different cyclic intermediates that can be obtained fromcyclization reaction from the intermediates from the core pathway aredepicted, as well as chemical transformation that lead from the cycliclactone intermediates to the various C5 compounds or building blocks.Additionally, different CoA intermediates that can be obtained from theintermediates in the core pathway are also represented. The pathway canbe used to produce levulinyl-CoA, from which either levulinic acid and4-valerolactone, or levulinate and/or other pentanoate esters such as4-oxo-pentanoate ester (compound P9) in FIG. 1, can readily be obtained.

FIG. 7 shows the principle of production of levulinic esters(levulinates) and levulinic acid from the levulinyl-CoA intermediatethrough the action of either a thioesterase or a transferase. The sidechain R can be any functional group such as, but not limited to methyl,ethyl, propyl, aryl, phenyl, naphthyl and other aromatic groups, as wellas alkyl group with oxygen and nitrogen substituents such as ketones,primary, secondary and tertiary alcohols, primary, secondary andtertiary amines, etc.

FIG. 8 shows the kinetic traces obtained when reacting two enoatereductase enzymes (Genbank accession numbers AAA64522 and AAD16106,labeled 6001 and 6002 in FIG. 8) with substrate 4-oxo-2-pentenoic acid(also known as acetylacrylic acid, see compound P2 in FIG. 1) andsubstrate cyclohexenone as a control. The curve labeled “6001 aceto” and“6002 aceto” show activity of the proteins in presence of 100 uM NADPHand the substrate 4-oxo-2-pentenoic acid. The curve labelled “6001cyclo” and “6002 cyclo” show the activity of the proteins in presence of100 uM NADPH and the substrate cyclohexenone. The decrease of absorptionat 340 nm, measuring the conversion of NADPH to the oxidized form NADP+,is monitored. This curve shows the conversion of the substrate4-oxo-2-pentenoic acid to levulinic acid (compound P1 in FIG. 1) by bothproteins. Control curves (labelled “6001 buffer” and “6002 buffer” and“buffer cyclo” and “buffer aceto”) show the decrease in absorbance withthe substrates alone in buffer or the proteins alone in buffer. Nosignificant activity is detected under these conditions. All curvesobtained in buffer Potassium Phosphate 100 mM, pH 7.0 and roomtemperature (25° C.). Initial NADPH concentration 100 uM,4-oxo-2-pentenoic acid initial concentration 100 mM, cyclohexenoneinitial concentration 50 mM. Protein concentration varied.

FIG. 9 demonstrates the activity of a class II aldolase enzyme fromPseudomonas Putida, HpaI aldolase (Genbank accession number ADA63518) onsubstrates acetaldehyde and pyruvate. Assay conditions were as follows:the protein was expressed and Ni-purified from E. Coli and reacted witha mix of acetaldehyde and pyruvate at an initial concentration of 100mg/ml, in Tris buffer, pH 8.0 supplemented with 100 mM MnCl₂. Theprotein and the substrates were incubated at room temperature for 30 minbefore quenching with HCl and run on an HPLC with an EPIC polar column.FIG. 9 shows the HPLC traces obtained, with the peaks corresponding tothe substrates and products indicated in the figure with the blackarrow. The chemical identity of the product, 4-hydroxy, 2-oxo pentanoicacid was confirmed by LC/MS (data not shown).

DETAILED DESCRIPTION OF THE INVENTION

In certain aspects and embodiments, the invention provides a chemicalpathway for the conversion of pyruvate obtained from sugars or othercarbon sources, to valuable C5 materials such as levulinic acid.Conceptually, the method of the invention provides a pathway that isorganized in at least two steps, and in some embodiments, from 4 to 8steps, such as 7 to 8 steps (see the core 8 steps depicted in FIG. 3),with up to 4 additional cyclization steps of intermediates obtainedalong the pathway. Attachment of the intermediate at multiple stages toa Co-enzyme A (CoA) moiety allows the pathway to lead to CoAintermediates such as levulinyl-CoA (see FIG. 6). In addition, fouroptional steps can lead to the cyclized variants of the keyintermediates in the pathway (see again FIG. 6).

According to various embodiments, a first step is glycolysis, whichconverts sugars (such as from biomass) to pyruvate, or alternatively anychemical conversion from sugars to pyruvate. A second step converts twomolecules of pyruvate into one molecule of 4-hydroxy 2-oxo-pentanoicacid and CO₂. An optional cyclization step produces the correspondinglactone, 2-oxo-4-valerolactone. An optional CoA attachment step can leadto 4-hydroxy-2-oxo pentanoyl-CoA. A third step reduces4-hydroxy-2-oxo-pentanoic acid into 2,4-dihydroxy-pentanoic acid, or4-hydroxy-2-oxo pentanoyl-CoA to 2,4-dihydroxy-pentanoyl-CoA, or2-oxo-4-valerolactone to 2-hydroxy-4-valerolactone. An optionalcyclization step produces the corresponding lactone,2-hydroxy-4-valerolactone, from either 2,4-dihydroxy-pentanoic acid or2,4-dihydroxy-pentanoyl-CoA. An optional CoA attachment step leads to2,4-dihydroxy pentanoyl-CoA from 2,4-dihydroxy pentanoic acid. A fourthstep oxidizes 2,4-dihydroxy-pentanoic acid to 2-hydroxy-4-oxo-pentanoicacid, or 2,4-dihydroxy-pentanoyl-CoA to 2-hydroxy-4-oxo-pentanoyl-CoA.An optional CoA attachment step converts 2-hydroxy-4-oxo-pentanoic acidto 2-hydroxy-4-oxo pentanoyl-CoA. A fifth step dehydrates2-hydroxy-4-oxo-pentanoic acid to 4-oxo-2-pentenoic acid, or2-hydroxy-4-oxo-pentanoyl-CoA to 4-oxo-2-pentenoyl-CoA. An optional CoAattachment step converts 4-oxo-2-pentenoic acid to4-oxo-2-pentenoyl-CoA. An optional step reduces further4-oxo-2-pentenoic acid to 4-hydroxy-2-pentenoic acid, or4-oxo-2-pentenoic acid to 4-hydroxy-2-pentenoyl-CoA, both of which canbe optionally cyclized to produce angelica lactone. Another optional CoAattachment step leads to 4-hydroxy-2-pentenoyl-CoA from4-hydroxy-2-pentenoic acid, which again can be optionally cyclized toproduce angelica lactone. An alternative embodiment of the invention“collapses” the fourth and fifth step into one single step. A sixth stepyields levulinic acid (4-hydroxy-pentanoic acid) through the reductionof 4-oxo-2-pentenoic acid in a similar manner as aboce. An optional stepattaches coenzyme A (CoA) to levulinic acid leading to levulinyl-CoA.Levulinyl-CoA can then be transformed into a variety of levulinic estersthrough the use of a transferase reacting with the appropriate alcohol.In some embodiments, a seventh step further reduces levulinic acid toproduce 4-hydroxy-pentanoic acid. An eighth step cyclizes4-hydroxy-pentanoic acid to yield 4-valerolactone.

In certain embodiments, steps 4 and 5 can be carried out in a singletransformation, an oxidative dehydration. In another embodiment of theinvention, steps 3 and 4 are reversed in order so that2-hydroxy-4-oxo-pentanoic acid is first oxidized into2,4-dioxo-pentanoic acid, and further reduced to2-oxo-4-hydroxy-pentanoic acid, so that the pathway of FIG. 3 becomesthe one represented in FIG. 4.

In another embodiment of the invention, steps 3, 5 and 6 (FIG. 3) arecarried out directly on the respective lactones L1, L2, L6, L7, L8, L9and L10, where the branching from linear intermediates producedinitially from pyruvate and acetaldehyde occurs at any one of thecyclization steps described in FIG. 6. This embodiment of the inventioncan be used either to obtain lactones directly, or, after hydrolysis, toobtain back any of the compounds P1 to P16 (including levulinic acid).

In yet another aspect of the invention, steps 2, 3, 4, 5 and 6 (FIG. 3)are carried out on the CoA intermediates, where the branching fromlinear intermediates produced initially from pyruvate and acetaldehydeoccur at any one of the CoA attachment steps described in paragraph FIG.6. This embodiment of the invention can be used to obtain any of thecompounds P1 to P16 (including levulinic acid) through the use ofthioesterases. Levulinic esters (P9) can be obtained from levulinyl-CoAand the appropriate alcohol by the use of a transferase.

Step 1: Conversion of Sugars to Pyruvate

The conversion of sugars to pyruvate is part of the well-studiedmetabolic pathway, glycolysis. In glycolysis, the action of multipleenzymes results in the conversion of each molecule of C6 sugar such asglucose to two molecules of pyruvate, two molecules of ATP and tworeducing equivalent in the form of two NAD(P)H molecules.

In one embodiment of the invention, pyruvate is obtained from glycolysisin a fermentation organism and subsequently used in the downstreampathway in the fermentation host. In an alternative embodiment, pyruvateis separated from the fermentation broth and subsequently processedaccording to the downstream pathway.

Step 2: Conversion of Pyruvate to 4-hydroxy-2-oxo-pentanoic Acid

4-hydroxy-2-oxo-pentanoic acid can be produced by the aldol addition ofacetaldehyde (an aldehyde) to pyruvate (an a keto-acid). The additionreacts one equivalent of acetaldehyde with one equivalent of pyruvate.Acetyladehyde can be obtained in various ways. For example, pyruvatedecarboxylase catalyzes the non-oxidative decarboxylation of pyruvate toacetaldehyde. Pyruvate decarboxylase from multiple eukaryotic orprokaryotic sources (e.g. Saccharomcyes cerevisiae) can therefore beused. In a preferred embodiment of the invention, acetyladehyde isproduced from pyruvate with the enzyme pyruvate decarboxylase.

Multiple aldolase have been isolated that have been shown to catalyzethe aldol addition between pyruvate and acetaldehyde. A class Ialdolase, 4-hydroxy-2-keto-pentanoic acid aldolase (HKP aldolase) is analdolase employing a Schiff base lysine and catalyzes the forward andreverse reaction. In one embodiment of the invention, the aldol additionbetween pyruvate and acetaldehyde is catalyzed by HKP aldolase from E.coli described in Pollard, J R et al., Substrate selectivity andbiochemical properties of 4-hydroxy-2-keto-pentanoic acid aldolase fromE. Coli, Appl. And Environ. Microbiology, 64(10):4093-4094 (1998), or ahomolog thereof, or mutants thereof (those mutants optionally beingobtained by protein engineering using computational design, directedevolution techniques or rational mutagenesis, or a combination thereof).Computational design techniques are disclosed in US 2009-0191607 and WO2010/077470, which are hereby incorporated by reference in its entirety.

There are at least two class II aldolases known to catalyze the additionbetween pyruvate and acetaldehyde, and two (BphI and HpaI) have beencharacterized in some level of detail in Wang W et al., Comparison oftwo metal-dependent pyruvate aldolases related by convergent evolution:substrate specificity, kinetic mechanism and substrate channeling,Biochemistry, 49:3774-3782 (2010). These enzymes employ a metalco-factor (either Zn or Mn are common). BphI and HpaI share nodetectable sequence similarity. Whereas BphI is stereoselective andleads to the 4S adduct, HpaI, due to its very open active site, producesa racemic mixture (4R and 4S adducts). BphI is allosterically coupled toBphJ, an acetaldehyde dehydrogenase, and is not active and stable whenexpressed in isolation. HpaI however, is expressable in E. coli byitself and shows activity. In an alternate embodiment of the invention,the aldol addition between pyruvate and acetaldehyde is catalyzed byHpaI or BphI, or mutants thereof (those mutants optionally beingobtained by protein engineering using computational design, directedevolution techniques or rational mutagenesis, or a combination of thethree).

As an extension, any suitable pyruvate aldolase and other similaraldolases (e.g. KDPG aldolase) catalyzing the aldol addition of analdehyde to a ketone can conceivably be reengineered to catalyze thealdol addition of acetaldehyde to pyruvate. The redesign may include,but is not limited to, achieving the desired substrate specificity forboth pyruvate and acetaldehyde, controlling the desiredstereoselectivity to produce either a racemic or enantiopure adducts((R)4-hydroxy-2-oxo-pentanoic acid and (S)4-hydroxy-3-oxo-pentanoicacid), stabilizing the enzyme to obtain the desired catalytic activityin the industrial conditions in which the invention is practiced (e.g.thermostabilization or stabilization in higher organic titer), and/orimproving the enzyme expressability and solubility in the context of theindustrial conditions in which the invention is practiced (e.g. in ametabolic pathway in Saccharomyces cerevisiae). In another embodiment ofthe invention, the aldol addition between pyruvate and acetaldehyde iscatalyzed by pyruvate aldolase, or any homologs and mutants thereof(those mutants optionally being obtained by protein engineering usingcomputational design, directed evolution techniques or rationalmutagenesis, or a combination of the three).

Finally, using the technique of de novo enzyme design such as the onedescribed in Zanghellini, A et al, New Algorithms and an in silicoBenchmark for Computational Enzyme Design, Protein Science 15:2785-2794(2006), it is possible to design new aldolase enzymes for substratesthat may or may not exist in nature. Up to 70 such aldolases have beendesigned de novo as described in US 2009-0191607, which is herebyincorporated by reference in its entirety. The application of thismethodology to the substrates pyruvate and acetaldehyde can lead toaldolases with the desired activity. In another embodiment of theinvention, the aldol addition between pyruvate and acetaldehyde iscatalyzed by a de novo designed aldolase.

Step 2′: Cyclization of 4-hydroxy-2-oxo-pentanoic Acid to2-oxo-4-valerolactone

4-hydroxy-2-oxo-pentanoic acid is cyclized into2-hydroxy-4-valerolactone (compound L7 in FIG. 1). In acidic to neutralsolutions, the thermodynamical equilibrium lies towards the cyclizationto the lactone. The cyclization to the lactone can be kineticallyenhanced by the use of either chemical or biochemical catalysis.Homogeneous and heterogeneous catalysts for lactonization include strongacid conditions (e.g. sulfuric acid), metal catalysts (e.g. palladium,rhubidium). Biochemical catalysis can be obtained by the action oflipases, esterases, proteases and lactonases under conditions that favorthe forward lactonization reaction (low to neutral pH/high organicsolvent titer) as demonstrated for example in Martin C H, et al,Integrated bioprocessing for pH-dependent of 4-valerolactone fromlevulinate in Pseudomonas Putida KT2440, Appl. and Environ, Microbiology76(2):417-424.

In one embodiment of the invention, 2-oxo-4-valerolactone is producedfrom 4-hydroxy-2-oxo-pentanoic acid, in the presence of a catalyst,after separation of 4-hydroxy-2-oxo-pentanoic acid from the fermentationbroth or cell-free solution. In another embodiment of the invention, thelactonization of 4-hydroxy-2-oxo-pentanoic acid to 2-oxo-4-valerolactoneis catalyzed directly by a lipase or esterase or protease or lactonase,or mutants thereof (those mutants being optionally obtained by proteinengineering using computational design, directed evolution techniques,rational mutagenesis, or a combination of the three).

Step 3: Reduction of 4-hydroxy-2-oxo-pentanoic Acid to2,4-dihydroxy-pentanoic Acid

Among the wide variety of natural dehydrogenases, in silico and/orexperimental screening can select dehydrogenases with substratespecificity that tolerates 4-hydroxy-2-oxo-pentanoic acid and2,4-dihydroxy-pentanoic acid. In addition, computational design,directed evolution techniques or rational mutagenesis, or a combinationof the three, can be used to alter or increase the substrate specificityof existing dehydrogenase towards 4-hydroxy-2-oxo-pentanoic acid and2,4-dihydroxy-pentanoic acid. Examples of suitable dehydrogenasestarting points include L- and D-lactate dehydrogenases (NAD(P)H- orHeme-dependent, from eukaryotic or bacterial origin), malate, aspartateand glutamate dehydrogenases (NAD(P)H-dependent from eukaryotic orbacterial origin), as well as alcohol dehydrogenases (such asNAD(P)H-dependent alkyl or phenyl alcohol dehydrogenases). Examples ofsuch dehydrogenases are listed in the example section.

In one embodiment of the invention, 4-hydroxy-2-oxo-pentanoic acid isselectively reduced to 2,4-dihydroxy-pentanoic acid using homogenous orheterogeneous chemical catalysis. 2,4-dihydroxy-pentanoic acid may ormay not be separated/purified from the fermentation or cell-freesolution to complete this step. Preferably, 2,4-dihydroxy-pentanoic acidis separated from the solution or fermentation broth before beingsubsequently subjected to said reduction.

In one embodiment of the invention, a NAD(P)H-dependent dehydrogenase isused to catalyze the reduction of the ketone at the 2 position in4-hydroxy-2-oxo-pentanoic acid. In another embodiment, saiddehydrogenase reduces the ketone with a high degree of substratespecificity for 4-hydroxy-2-oxo pentanoic acid and high regioselectivelyfor the ketone at the 2 position. In one embodiment of the invention,said dehydrogenase is not stereoselective and can accept both 4R and 4Senantiomers. In another embodiment of the invention, said dehydrogenasereduces selectively either the 4R or 4S enantiomeric form of4-hydroxy-2-oxo-pentanoic acid.

In another embodiment of the invention, a FAD-dependent dehydrogenase isused instead of a NAD(P)H-dependent dehydrogenase, preferably with ahigh degree of substrate and regioselectivity. In one embodiment of theinvention, said dehydrogenase is not stereoselective and can accept both4R and 4S enantiomers. In another embodiment of the invention, saiddehydrogenase reduces selectively either the 4R or 4S enantiomeric formof 4-hydroxy-2-oxo-pentanoic acid.

In another embodiment of the invention, a FMN-dependent dehydrogenase isused instead of a NAD(P)H-dependent dehydrogenase, preferably with ahigh degree of substrate and regioselectivity. In one embodiment of theinvention, said dehydrogenase is not stereoselective and can accept both4R and 4S enantiomers. In another embodiment of the invention, saiddehydrogenase reduces selectively either the 4R or 4S enantiomeric formof 4-hydroxy-2-oxo-pentanoic acid.

In yet another embodiment of the invention, a ferricytochrome-dependentdehydrogenase is used instead of a NAD(P)H-dependent dehydrogenase,preferably with a high degree of substrate and regioselectivity. In oneembodiment of the invention, said dehydrogenase is not stereoselectiveand can accept both 4R and 4S enantiomers. In another embodiment of theinvention, said dehydrogenase reduces selectively either the 4R or 4Senantiomeric form of 4-hydroxy-2-oxo-pentanoic acid.

In yet another embodiment of the invention, a quinone-dependantdehydrogenase is used instead of a NAD(P)H-dependent dehydrogenase,preferably with a high degree of substrate and regioselectivity. In oneembodiment of the invention, said dehydrogenase is not stereoselectiveand can accept both 4R and 4S enantiomers. In another embodiment of theinvention, said dehydrogenase reduces selectively either the 4R or 4Senantiomeric form of 4-hydroxy-2-oxo-pentanoic acid.

Step 3′: Cyclization of 2,4-dihydroxy-pentanoic Acid to 2-hydroxy4-valerolactone

2,4-dihydroxy-pentanoic acid is cyclized into 2-hydroxy-4-valerolactone(compound L6 in FIG. 1). In acidic to neutral solutions, thethermodynamical equilibrium lies towards the cyclization to4-valerolactone. The same remarks about thermodynamic equilibrium andchemical and biochemical catalysis hold as described above.

In one embodiment of the invention, 2-hydroxy-4-valerolactone isproduced from 2,4-dihydroxy-pentanoic acid, in the presence of acatalyst, after separation of 2,4-dihydroxy-pentanoic acid from thefermentation broth or cell-free solution. In another embodiment of theinvention, the lactonization of 2,4-dihydroxy-pentanoic acid to2-hydroxy-4-valerolactone is catalyzed directly by a lipase or esteraseor protease or lactonase, or mutants thereof (those mutants beingobtained by protein engineering using computational design, directedevolution techniques or rational mutagenesis, or a combination of thethree).

Step 4: Oxidation of 2,4-dihydroxy-pentanoic Acid to4-oxo-2-hydroxy-pentanoic Acid

In one embodiment of the invention, 2,4-dihydroxy-pentanoic acid isselectively oxidized to 4-oxo-2-hydroxy-pentanoic acid using homogenousor heterogeneous chemical catalysis. 2,4-dihydroxy-pentanoic acid may ormay not be separated/purified from the fermentation or cell-freesolution to complete this step. Preferably, 4-oxo-2-hydroxy-pentanoicacid is separated from the solution or fermentation broth before beingsubsequently subjected to said oxidation.

In a preferred embodiment of the invention, an NAD(P)H-dependentdehydrogenase is used to catalyze the oxidation of the hydroxyl at the 4position in 2,4-dihydroxy-pentanoic acid. In a preferred embodiment,said dehydrogenase oxidizes the hydroxyl with a high degree of substratespecificity for 2,4-dihydroxy pentanoic acid and high regioselectivelyfor the hydroxyl at the 4 position. Preferably, said dehydrogenaseaccepts the four different enantiomers (2R4R, 2R4S, 2S4R, 2S4S) of2,4-dihydroxy pentanoic acid. In an alternative embodiment, saiddehydrogenase is oxidizing selectively either the 2R (2R4R, 2R4S) or 2S(2S4R,2S4S) enantiomers of 2,4-dihydroxy-pentanoic acid, whichever isthe most abundant enantiomer resulting from the previous reduction of4-hydroxy-2-oxo-pentanoic acid. Examples of such dehydrogenases arelisted in the example section.

In another embodiment of the invention, a FAD-dependent dehydrogenase isused to catalyze the oxidation of the hydroxyl at the 4 position in2,4-dihydroxy-pentanoic acid. In a preferred embodiment, saiddehydrogenase oxidizes the hydroxyl with a high degree of substratespecificity for 2,4-dihydroxy-pentanoic acid and high regioselectivelyfor the hydroxyl at the 4 position. Preferably, said dehydrogenaseaccepts the four different enantiomers (2R4R, 2R4S, 2S4R, 2S4S) of2,4-dihydroxy-pentanoic acid. In a alternative embodiment, saiddehydrogenase is oxidizing selectively either the 2R (2R4R, 2R4S) or 2S(2S4R, 2S4S) enantiomers of 2,4-dihydroxy-pentanoic acid, whichever isthe most abundant enantiomer resulting from the reduction of4-hydroxy-2-oxo-pentanoic acid.

In another embodiment of the invention, a FMN-dependent dehydrogenase isused to catalyze the oxidation of the hydroxyl at the 4 position in2,4-dihydroxy-pentanoic acid. In a preferred embodiment, saiddehydrogenase oxidizes the hydroxyl with a high degree of substratespecificity for 2,4-dihydroxy-pentanoic acid and high regioselectivelyfor the hydroxyl at the 4 position. Preferably, said dehydrogenaseaccepts the four different enantiomers (2R4R, 2R4S, 2S4R, 2S4S) of2,4-dihydroxy-pentanoic acid. In a alternative embodiment, saiddehydrogenase is oxidizing selectively either the 2R (2R4R, 2R4S) or 2S(2S4R,2S4S) enantiomers of 2,4-dihydroxy-pentanoic acid, whichever isthe most abundant enantiomer resulting from the reduction of4-hydroxy-2-oxo-pentanoic acid.

In yet another embodiment of the invention, a ferricytochrome-dependentdehydrogenase is used to catalyze the oxidation of the hydroxyl at the 4position in 2,4-dihydroxy-pentanoic acid. In a preferred embodiment,said dehydrogenase oxidizes the hydroxyl with a high degree of substratespecificity for 2,4-dihydroxy-pentanoic acid and high regioselectivelyfor the hydroxyl at the 4 position. Preferably, said dehydrogenaseaccepts the four different enantiomers (2R4R, 2R4S, 2S4R, 2S4S) of2,4-dihydroxy-pentanoic acid. In an alternative embodiment, saiddehydrogenase is oxidizing selectively either the 2R (2R4R, 2R4S) or 2S(2S4R, 2S4S) enantiomers of 2,4-dihydroxy-pentanoic acid, whichever isthe most abundant enantiomer resulting from the reduction of4-hydroxy-2-oxo-pentanoic acid.

In yet another embodiment of the invention, a quinone-dependentdehydrogenase is used to catalyze the oxidation of the hydroxyl at the 4position in 2,4-dihydroxy-pentanoic acid. In a preferred embodiment,said dehydrogenase oxidizes the hydroxyl with a high degree of substratespecificity for 2,4-dihydroxy pentanoic acid and high regioselectivelyfor the hydroxyl at the 4 position. Preferably, said dehydrogenaseaccepts the four different enantiomers (2R4R, 2R4S, 2S4R, 2S4S) of2,4-dihydroxy-pentanoic acid. In a alternative embodiment, saiddehydrogenase is oxidizing selectively either the 2R (2R4R, 2R4S) or 2S(2S4R,2S4S) enantiomers of 2,4-dihydroxy-pentanoic acid, whichever isthe most abundant enantiomer resulting from the reduction of4-hydroxy-2-oxo-pentanoic acid.

Step 5: Dehydration of 4-oxo-2-hydroxy-pentanoic Acid to4-oxo-2-pentenoic Acid

Classically, chemical dehydration is achieved with either homogeneous orheterogeneous catalysis, such as temperature >100° C., concentrated acid(4.0M sulfuric acid) and/or metal oxide catalyst (zinc or aluminiumoxides). In one embodiment of the invention, 4-oxo-2-hydroxy-pentanoicacid obtained after the reduction and oxidation steps is dehydratedchemically to 4-oxo-2-pentenoic acid by homogeneous or heterogeneouscatalysis. 4-oxo-2-hydroxy-pentanoic acid may or may not beseparated/purified from the fermentation or cell-free solution tocomplete this step. Preferably, 4-oxo-2-hydroxy-pentanoic acid isseparated from the solution or fermentation broth before being subjectedto said dehydration.

The dehydration of organic compounds can alternatively be catalyzed by adehydratase enzyme. Several classes of dehydratase have beencharacterized and rely on different mechanisms: radical based mechanismsuch as in vitamin B12-dependent or SAM-dependent dehydratases (e.g.diol dehydratase, glycerol dehydratase), Lewis-acid mechanism suchIron-Sulfur containing dehydratases (e.g. dihydroxy-acid dehydratase,aconitase) and enolate ion intermediate mechanism such as diaciddehydratase (e.g. tartrate dehydratase). Whereas all mechanisms areapplicable to the dehydration of 4-oxo-2-hydroxy-pentanoic acid,mechanisms relying on an enolate intermediate are preferred because theformation of an enolate anion on the carbonyl 13 to the hydroxyl beingeliminated lowers the pKa of the α-proton, thereby allowing it to bereadily abstracted by a general acid/base group. An additional generalacid/base group protonates the leaving water molecule. This mechanism isexploited by a wide variety of natural dehydratases: Magnesium-dependentdehydratases from the enolase superfamily, such as tartrate dehydratase,gluconate dehydratase, use this mechanism for the dehydration ofstructurally diverse diacids with high substrate specificity, asdescribed for instance in Gerlt et al., Divergent evolution in theenolase superfamily: the interplay of mechanism and specificity,Biochemistry, 433:59-70 (2005). Fumarase (also known as fumaratehydratase) catalyzes the enolate-based reversible hydration of malate tofumarate. Enoyl dehydratase (also known as crotonase) uses the enolateanion of a CoA thioester to catalyze the reversible hydration of variousCoA substrates (see for instance Holden et al., The CrotonaseSuperfamily: divergently related enzymes that catalyze differentreactions involving acyl Coenzyme A thioesters, Acc. Chem. Res.34:145-157. (2001))

In one embodiment of the invention, the dehydration of4-oxo-2-hydroxy-pentanoic acid to 4-oxo-2-pentenoic acid is catalyzed bya dehydratase. In a preferred embodiment of the invention, saiddehydratase uses an enolate intermediate to catalyze the dehydration.Preferably, said dehydratase is a member of the enolase superfamily,fumarase or enoyl-coA dehydratase superfamilies, or mutants thereofobtained by protein engineering. In a preferred embodiment of theinvention, said dehydratase exhibits a high level of substratespecificity for 4-oxo-2-hydroxy-pentanoic acid. In another preferredembodiment of the invention, said dehydratase dehydrates equally the 2Rand 2S enantiomers of 4-oxo-2-hydroxy-pentanoic acid. In an alternativeembodiment of the invention, said dehydratase dehydrates selectivelyeither the 2R or 2S enantiomer of 4-oxo-2-hydroxy-pentanoic acid.

Alternative to Step 4 and 5:

Alternatively to a 2-step conversion of 2,4-dihydroxy-pentanoic acid to4-oxo-2-pentenoic acid, a 1-step conversion can be achieved using anoxidative dehydration. Oxidative dehydrations are common in themetabolism of sugars. The so-called 4,6 dehydratase enzymes, such asUDP-GlcNAc-inverting 4,6-dehydratase which structural details aredescribed in Ishiyama et al., Structural studies of FlaA1 fromhelicobacter pylori reveal the mechanism for inverting 4,6-dehydrataseactivity, J. Bio. Chem. 281(34):24489-24495 (2006). In one embodiment ofthe invention, such a 4,6-dehydratase is used to catalyze the oxidativedehydration of 2,4-dihydroxy-pentanoic acid to 4-oxo-2-pentanoic acid.In one aspect of the invention, said 4,6-dehydratase is enantioselectiveand dehydrates preferably one of the enantiomers of2,4-dihydroxy-pentanoic acid (either 2R4R, 2R4S, 2S4R or 2S4S). Inanother aspect of the invention, said 4,6-dehydratase is notenantioselective and dehydrates with similar catalytic efficiency two ormore of the enantiomers of 2,4-dihydroxy-pentanoic acid. In a preferredembodiment of the invention, the 4,6-dehydratase is highly active on2,4-dihydroxy-pentanoic acid is obtained from a natural 4,6-dehydrataseby protein engineering using computational design, directed evolutiontechniques or rational mutagenesis, or a combination thereof.

Step 6: Reduction of 4-oxo-2-pentenoic Acid to 4-oxo-pentanoic Acid(Levulinic Acid)

Double bonds on substituted alkenes can be reduced (hydrogenated) toobtain the corresponding saturated alkanes. Substituted alkenes can bereduced using chemical catalysis or, generally asymmetrically, usingbiocatalysts such as enoate reductases as reviewed in Stuermer et al.,Asymmetric bioreduction of activated C═C bonds using enoate reductasesfrom the old yellow enzyme family, Curr. Opin. In Chem. Bio. 11:203-213(2007). Enoate reductases have been characterized from both eukaryotic,such as Sacharomyces cerevisiae and Marchantia and prokaryoticorganisms, such as Clostridium. The family of enoate reductase enzymesis dependent on a flavin cofactor (FMN) that gets oxidized at eachturnover of the enzyme. Except for one known case, which isnicotinamide-independent, the flavin cofactor is in turned reduced by anicotinamide cofactor, either NADH or NADPH, that also binds in theactive site. Upon completion of one turnover, the substrate has beenreduced whereas the cofactor NAD(P)H has been oxidized to NAD(P)+.Enoate reductases differ in their substrate specificity. However,several enoate reductases such as yeast and Clostridium enoatereductases have a broad substrate specificity and can accommodate linearsubstituted alkenes (with acids or ketone functional groups) as well assubstituted lactones such as 4-valerolactone.

In one embodiment of the invention, 4-hydroxy-2-oxo-pentanoic acid isseparated from the separation broth or cell-free solution and the doublebond selectively reduced using homogenous or heterogeneous catalysis.

In another embodiment of the invention, an enoate reductase enzyme isused to reduce 4-hydroxy-2-oxo-pentanoic acid into levulinic acid. In apreferred embodiment, said enoate reductase is dependent on both FMNH2and NAD(P)H cofactors, said NAD(P)H cofactor being used in the activesite to regenerate FMNH2 to its oxidoreduction state before catalysis.In a preferred embodiment of the invention, said enoate reductase iscloned and expressed in the fermentation host. In a alternativeembodiment, said enoate reductase is used extracellularly, or in acell-free system with an adequate cofactor regeneration system. Inanother alternative embodiment, said reduction is catalyzed by a wholecell catalyst expressing one or several enoate reductases, such thatsaid cell is different from the fermentation host cell(s) in which partor the totality of the pathway is used.

Step 7: Reduction of 4-oxo-pentanoic Acid (Levulinic Acid) to4-hydroxy-pentanoic Acid

Similarly to step 3 (paragraphs [0037] to [0043]), the reduction of theketone at the 4 position on levulinic acid can be achieved either bychemical catalysis means or by the use of a dehydrogenase biocatalyst.In the context of a metabolic pathway, this last reduction (andcorresponding oxidation of one reducing equivalent) ensures the redoxbalance of the whole pathway from C5 and/or C6 sugars.

In one embodiment of the invention, levulinic acid is separated from thebroth or cell-free solution and the ketone at the 4 positions isselectively reduced using homogenous or heterogeneous catalysis to yield4-hydroxy-pentanoic acid.

In an alternate embodiment of the invention, an NAD(P)-dependentdehydrogenase is used to catalyze the reduction of the ketone at the 4position on levulinic acid to the corresponding hydroxyl to yield4-hydroxy-pentanoic acid. In a preferred embodiment, said dehydrogenasereduces the ketone with a high degree of substrate specificity forlevulinic acid and high regioselectively for the ketone at the 4position. Preferably, said dehydrogenase is the same enzyme as for theoxidation of the hydroxyl at the 4 position of 4-oxo-2-hydroxy-pentanoicacid, or a mutant thereof (the mutant being obtained by computationaldesign or experimental mutagenesis, or a combination of the two). In apreferred embodiment of the invention, said dehydrogenase producesselectively one of the enantiomers (4R or 4S) of 4-hydroxy-pentanoicacid. In a alternative embodiment, said dehydrogenase produces a racemicmixture of the 4R and 4S enantiomers of 4-hydroxy-pentanoic acid.

Step 8: Cyclization of 4-hydroxy-pentanoic Acid to 4-valerolactone

4-hydroxy-pentanoic acid is cyclized into 4-valerolactone (also known asγ-valerolactone, compound L1 in FIG. 1). In acidic solutions, thethermodynamical equilibrium lies towards the cyclization to4-valerolactone. The same remarks about thermodynamic equilibrium andchemical and biochemical catalysis hold as in paragraph [0035].

In one embodiment of the invention, 4-valerolactone is produced from4-hydroxy-pentanoic acid, in the presence of a catalyst, afterseparation of 4-hydroxy-pentanoic acid from the fermentation broth orcell-free solution. In a preferred embodiment of the invention, anenantiopure 4-hydroxy-pentanoic acid (either the 4R or 4S enantiomer) isconverted by said catalyst into the enantiopure 4-valerolactone. In analternative embodiment, a racemic mixture of the two enantiomers for4-hydroxy-pentanoic acid (4R and 4S) is converted by said catalyst intoa racemic mixture of 4-valerolactone.

In another embodiment of the invention, the lactonization of4-hydroxy-pentanoic acid to 4-valerolactone is catalyzed directly by alipase or esterase or protease or lactonase, or mutants thereof (thosemutants being obtained by protein engineering using computationaldesign, directed evolution techniques or rational mutagenesis, or acombination of the three) within a cell or outside of a cell. In apreferred embodiment of the invention, said lipase or esterase orprotease or lactonase acts on the enantiopure 4-hydroxy-pentanoic acidsubstrate to yield an enantiopure 4-valerolactone. In a alternativeembodiment, said lipase or esterase of protease or lactonase acts on aracemic mixture of the 4R and 4S enantiomers of 4-hydroxy-pentanoic acidto yield a racemic mixture of the 4R and 4S enantiomer of4-valeralactone.

Examples

Examples of Pyruvate Decarboxylases Enzymes:

an enzyme of the pyruvate decarboxylase family (EC number EC 4.1.1.1)such as pyruvate decarboxylase enzyme can be used to catalyze the firststep of the pathway, the conversion of pyruvate to acetaldehyde. Table 1below lists examples of such enzymes (along with their sourceorganisms), that have been studied and characterized in the literature,with their accession number for the public database GenBank (NCBI)listed. Homologous enzymes, for instance protein and DNA sequencesobtained from the sequences in table 1 (or their reverse translation)using an alignment software such as, but not limited to, Blast,PSI-Blast or HMMER3, and with an alignment e-value<0.1, can also beused.

TABLE 1 GenBank (protein) Accession Number Organism CAA39398Saccharomyces Cerevisiae AAM21208 Acetobacter pasteurianus NP_195033Arabidopsis thaliana AAA20440 Aspergillus parasiticus EEQ44875 Candidaalbicans AAN77243 Candida glabrata XP_002549529 Candida tropicalis XP001703530 Chlamydomonas reinhardtii AAZ05069 Citrus Sinensis ADZ22807Clostridium acetobytulicun YP_003531827 Erwinia amylovora AAG13131Fragaria × ananassa AAA85103 Hanseniaspora uvarum CAA59953 Kluyveromyceslactis AAA35267 Kluyveromyces marxianus AAP75899 Lachancea kluyveriAAS49166 Lactococcus lactis AAA33567 Neurospora crassa BAC20138 Oryzasativa AAX33300 (1) and AAX33299 Petunia × hybrida BAI23188 Pichiajadinii CAA91444 Pisum sativum ABU96175 Populus tremula × Populus albaABZ79223 Prunus armeniaca AAM73539 (A) and AAM73540 (B) Rhizopus oryzaeACM04215 Rhodobacter sphaeroides NP_948455 Rhodopseudomonas palustrisAAL18557 Sarcina ventriculi AAC03164 (1) and AAC03165 (2)Scheffersomyces stipitis CAA90807 Schizosaccharomyces pombe BAC23043Solanum tuberosum AAG22488 Vitis vinifera CAH56494 Wickerhamomycesanomalus CAG80835 Yarrowia lipolytica NP 001105645 Zea mays CAB65554Zygosaccharomyces bisporus AAM49566 Zymobacter palmae CAA42157 Zymomonasmobilis

Examples of Aldolase Enzymes Catalyzing the Production of4-Hydroxy-2-Keto-Pentanoic Acid:

Homologous enzymes, for instance protein and DNA sequences obtained fromthe sequences in tables below (or their reverse translation) using analignment software such as, but not limited to, Blast, PSI-Blast orHMMER3, and with an alignment e-value<0.1, can also be used.

TABLE 2 class I aldolase: EC 4.1.3.39 official name: 4-hydroxy-2-oxovalerate aldolase GenBank (protein) Accession Number OrganismP51020 Escherichia Coli

TABLE 3 class II aldolases: EC 4.1.3.39 official name: 4-hydroxy-2-oxovalerate aldolase GenBank (protein) Accession Number OrganismADA63518 Pseudomonas putida ABE37049 Burkholderia Xenovorans

TABLE 4 examples of additional pyruvate aldolases susceptible tocatalyze the reaction, either as WT or after protein engineering:GenBank (protein) EC Accession Number number Name Organism Q79EM84.1.2.34 4-(2-carboxyphenyl)-2-oxobut-3- Nocardioides sp. enoatealdolase Q51947 4.1.2.45 Trans-o- Pseudomonas Putidahydroxybenzylidenepyruvate hydratase-aldolase NP_746573 4.1.3.174-hydroxy-4-methyl-2-oxoglutarate Pseudomonas Putida aldolase

Examples of Dehydrogenase Enzymes Able to Reduce the Ketone at Position4 of Pentanoic Acid Derivatives to a Secondary Alcohol(Hydroxyl)/Oxidize a Secondary Alcohol (Hydroxyl) at Position 4 ofPentanoic Acid Derivatives to a Ketone:

Homologous enzymes, for instance protein and DNA sequences obtained fromthe sequences in tables below (or their reverse translation) using analignment software such as, but not limited to, Blast, PSI-Blast orHMMER3, and with an alignment e-value<0.1, can also be used.

A wide variety of dehydrogenases are capable of oxidizing/reducingsecondary alcohols/ketons, with various degrees of substratespecificity. The dehydrogenase sequences listed below ares some examplesof dehydrogenases reported in the literature to be active on secondaryalcohols/ketones substituents on alkyl chains of three carbons or more.

TABLE 5 GenBank (protein) EC Accession Number number Name OrganismCAA09258 1.1.1.1 Medium-chain and short-chain Sulfolobus solfataricussecondary alcohol dehydrogenase CAA99098 1.1.1.B3* (S)-specificsecondary alcohol Saccharomyces cerevisiae dehydrogenase AAA344081.1.1.B4* (R)-specific secondary alcohol Saccharomyces cerevisiaedehydrogenase Q56840 1.1.1.268 2-(R)-hydroxypropyl-CoM Xanthobacterautotrophicus dehydrogenase Q56841 1.1.1.269 2-(S)-hydroxypropyl-CoMXanthobacter autotrophicus dehydrogenase ADX68565 1.1.1.211long-chain-3-hydroxyacyl-CoA Weeksella virosa dehydrogenase AAK181671.1.1.35 3-hydroxacyl-CoA dehydrogenase Pseudomonas putida YP_0043669171.1.1.178 3-hydroxy-2-methylbutyryl-CoA Marinithermus dehydrogenasehydrothermalis NP_062043 1.1.1.184 carbonyl reductase Rattus norvegicus*temporary (non-official) EC numbers assigned by enzyme database BRENDA

Examples of Dehydrogenase Enzymes to Reduce 2,4-Dioxo Pentanoic Acid to4-Oxo-2-Hydroxy-Pentanoic Acid:

Homologous enzymes, for instance protein and DNA sequences obtained fromthe sequences in tables below (or their reverse translation) using analignment software such as, but not limited to, Blast, PSI-Blast orHMMER3, and with an alignment e-value<0.1, can also be used.

Lactate dehydrogenase enzymes with broad substrate specificitydemonstrated in the literature to accept the substrate 2,4-dioxopentanoic acid. The two sequences below have differentstereoselectivities.

TABLE 6 GenBank (protein) EC Accession Number number Name Organism2LDB_A 1.1.1.27 L-Lactacte Bacillus dehydrogenase StearothermophilusQ5HLA0 1.1.1.28 D-Lactate Staphylococcus dehydrogenase epidermidis

Example of Dehydratase Enzymes Catalyzing the Conversion of4-Oxo-2-Hydroxy-Pentanoic Acid to 4-Oxo-2-Pentenoic Acid:

Homologous enzymes, for instance protein and DNA sequences obtained fromthe sequences in tables below (or their reverse translation) using analignment software such as, but not limited to, Blast, PSI-Blast orHMMER3, and with an alignment e-value<0.1, can also be used.

Dehydratases of the Enolate Superfamily:

these dehydratase enzymes, which are structurally related to the“enolase” family of enzymes, stabilize the enolate ion formed afterabstraction of one of the hydrogen a to the acid functional group.Because these enzymes rely on the stabilization of the enolate anion todecrease the activation energy for the dehydration reaction, they can beactive on substrate with the hydroxyl to be eliminated β with either acarboxylic acid, ketone or ester functional groups. Several examples ofthis class of dehydratase is provided in the table below:

TABLE 7 GenBank (protein) EC Accession Number number Name Organism2HXT_A 4.2.1.68 L-fuconate dehdyratase Xanthomonas Campestris ACT447364.2.1.32 L-tartrate dehydratase Escherichia Coli 2DW7_A 4.2.1.81D-tartrate dehydratase Bradyrhizobium Japonicum 2I5Q_A 4.2.1.90L-rhamnonate dehydratase Escherichia Coli YP_003470410 4.2.1.39gluconate dehydratase Staphylococcus lugdunensis YP_001461084 4.2.1.8D-mannonate dehydratase Escherichia Coli EGP22937 4.2.1.6 D-galactonatedehydratase Escherichia Coli

Dehydratases of the Enoyl-coA Hydratase, or “Crotonase”, Family:

these enzymes can catalyze the reversible addition/elimination of awater molecule to/from a α,β unsaturated thio-esters (coenzyme Aderivatives). Because they rely on stabilization of the enolate anionformed after proton abstraction, the enzymes are also able to catalyzethe hydration (and reversible dehydration) of α,β unsaturated carboxylicacids and ketones. Contrary to the dehydratase from the enolasesuperfamily, these enzymes do not require any cofactor.

TABLE 8 GenBank (protein) EC Accession Number number Name OrganismEGI23865 4.2.1.55 3-hydroxybutyryl-CoA dehydratase Escherichia ColiYP_001730392 4.2.1.17 enoyl-CoA hydratase Escherichia Coli 1DUB_A4.2.1.74 Long-chain enoyl-CoA hydratase Rattus Norvegicus YP_0030226134.2.1.100 cyclohexa-1,5-dienecarbonyl-CoA Geobacter sp. M21 hydrataseACL95949 4.2.1.101 trans-feruloyl-CoA hydratase Caulobacter CrescentusYP_003394145 4.2.1.107 3alpha,7alpha,12alpha-trihydroxy- Conexibacterwoesei 5beta-cholest-24-enoyl-CoA hydratase AEE35803 4.2.1.119 enoyl-CoAhydratase 2 Arabidopsis thaliana

Dehydratases of the Fumarase C Family (Enzymes of the Family Fumarase Aand B Use an Iron-Sulfur Cluster):

As for the enoyl-coA hydratases family, these enzymes stabilize theenolate without requiring any cofactor. Substrate binding and transitionstate stabilization is achieved with active site amino-acids.

TABLE 9 GenBank (protein) EC Accession Number number Name OrganismACI83235 4.2.1.2 fumarate hydratase Escherichia Coli

Other Dehydratases:

all other known dehydratases (EC numbers 4.2.1.*) may also be used tocatalyze the dehydration of 4-oxo-2-hydroxy pentanoic acid to4-oxo-2-pentenoic acid, such as a dehydratase enzymes relying on anIron-Sulfur cluster (e.g. dihydroxy-diol dehydratase, fumarase A and C)or vitamin B12-dependent and SAM-dependent dehydratases such as glyceroland propanediol dehydratase.

Examples of Oxidase/Epimerase Enzymes Capable of Catalyzing theOxidative Dehydration/Conversion of 2,4-Dihydroxy-Pentanoic Acid to4-Oxo-2-Pentenoic Acid:

Homologous enzymes, for instance protein and DNA sequences obtained fromthe sequences in tables below (or their reverse translation) using analignment software such as, but not limited to, Blast, PSI-Blast orHMMER3, and with an alignment e-value<0.1, can also be used.

TABLE 10 GenBank (protein) EC Accession Number number Name OrganismZP_01202902 4.2.1.115 UDP-N-acetyl- Flavobacteria glucosamine 4,6-bacterium dehydratase

Examples of Enzymes Catalyzing the Reduction of 4-Oxo, 2-HydroxoPentanoic Acid to Levulinic Acid:

Homologous enzymes, for instance protein and DNA sequences obtained fromthe sequences in tables below (or their reverse translation) using analignment software such as, but not limited to, Blast, PSI-Blast orHMMER3, and with an alignment e-value<0.1, can also be used.

The family of enzymes called enoate-reductases, or more informally OldYellow Enzymes, are NAD(P)H and FMN dependent enzyme catalyzing thereversible reduction of α,β unsaturated thioesters, carboxylic acids andketones. They exhibit broad substrate specificities and the followingsequences have been successfully proved experimentally (see data) tocatalyze the reduction of 4-oxo, 3-hydroxy pentanoic acid to levulinicacid.

TABLE 11 GenBank (protein) EC Accession Number number Name OrganismAAA64522 1.3.1.31 Old Yellow Enzyme 1 Saccharomyces Cerevisiae AAD161061.3.1.31 2-cyclohexen-1-one Pseudomonas reductase Ncr syringaeMultiple point mutants of the enzyme NCR from Pseudomonas syringae havealso been shown experimentally to exhibit various catalytic activitiestoward 4-oxo, 2-hydroxo pentanoic acid as a substrate. These mutantscorrespond to Y178A, P242Q, D338Y and F315Y in the amino-acid numberingof sequence AAD16106.

Examples of Enzymes Able to Catalyze the Lactonization of 4-HydroxyAcids into their Corresponding Cyclic Esters (Lactones):

Homologous enzymes, for instance protein and DNA sequences obtained fromthe sequences in tables below (or their reverse translation) using analignment software such as, but not limited to, Blast, PSI-Blast orHMMER3, and with an alignment e-value<0.1, can also be used.

Many kinds of lactonases (e.g. lactonohydrolases) are known that can beused to catalyze the reversible formation of 1,4 cyclic esters from4-hydroxy acids. In particular, 1.4 lactonases (EC 3.1.1.25) show somespecificity towards 4-hydroxy acids and are therefore sequences ofchoice to catalyze the reactions of steps 8 in FIGS. 3,4 and 5, and themultiple lactonization reactions in FIG. 6. Particularly, some1,4-lactonases have been assayed with 4-hydroxy pentanoic acid andreported to catalyze its reversible cyclization intogamma-valerolactone. The table below lists some lactonase enzymes thathave been reported in the literature to catalyze this reaction.

TABLE 12 GenBank (protein) EC Accession Number number Name OrganismYP_001903921 3.1.1.25 1,4 lactonase Xanthomonas campestris AAB418353.1.1.17 Paraoxonase 1 (PON1)/ Homo Sapiens gluconolactonase

A wide variety of other characterized lactonases are susceptible tocatalyze the cyclization of 4-hydroxy acids. Below is a table that liststhe EC numbers corresponding to existing lactonases (a subclass ofcarboxyesterases).

TABLE 13 EC number Name 3.1.1.15 L-arabinolactonase 3.1.1.17gluconolactonase 3.1.1.19 uronolactonase 3.1.1.24 3-oxoadipateenol-lactonase 3.1.1.25 1,4-lactonase 3.1.1.27 4-pyridoxolactonase3.1.1.30 D-arabinolactonase 3.1.1.31 6-phosphogluconolactonase 3.1.1.36limonin-D-ring lactonase 3.1.1.37 steroid-lactonase 3.1.1.38Triacetate-lactonase 3.1.1.39 actinomycin lactonase 3.1.1.46deoxylimonate A-ring-lactonase 3.1.1.57 2-pyrone-4,6-dicarboxylatelactonase 3.1.1.65 L-rhamnono-1,4-lactonase 3.1.1.68xylono-1,4-lactonase 3.1.1.81 quorum-quenching N-acyl- homoserinelactone

Finally esterases, lipases and peptidases/amidases have been observed tocatalyze lactonization reaction under appropriate experimentalconditions (non-alkaline pH and usually room temperature. For example,lipases are referenced in PCT/US2010/055524 for lactonization andamidase/peptidase have been used successfully to synthetize lactones inWO/2009/142489, both of which are hereby incorporated by reference.

Examples of Non-Biocatalytic Methods to Catalyze the Lactonization of4-Hydroxy Acids into their Corresponding Cyclic Esters (Lactones):

there are multiple non-biocatalytic ways to catalyze the1,4-lactonization of hydroxacids. For instance, it is well-known thatsuch lactonization is acid-catalyzed and therefore lowering the pH ofthe medium (whether inside or outside of living cells) increases therate of the lactonization reaction. Additionally, it has been reportedin PCT/US2010/055524 (which is hereby incorporated by reference) thatactivation through group transfer on the acid functional group of the4-hydroxy acid is sufficient, under reasonable conditions such as pH 2.5to 7.0 and room temperature, to yield the lactone form quantitatively.For instance, PCT/US2010/055524 lists (1) activation with a phosphategroup (by producing in this case 4-hydroxylbutyryl phosphate) and (2)activation with coenzyme A (by producing 4-hydroxylbutyryl-CoA).Synthesis of the intermediates 4-hydroxylpentanoyl-phosphate or4-hydroxylpentanoyl-CoA, using a natural or engineered kinase enzyme orCoA synthetase respectively, or chemical synthesis, is expected toresult in similar activation and spontaneous lactonization underappropriate conditions.

All references cited herein are hereby incorporated by reference for allpurposes.

1.-58. (canceled)
 59. A method for producing 2-oxo-4-valerolactone, themethod comprising: converting pyruvate to 4-hydroxy-2-oxo-pentanoic acidby aldol addition, and converting 4-hydroxy-2-oxo-pentanoic acid to2-oxo-4-valerolactone through lactonization.
 60. The method of claim 59,wherein the 2-oxo-4-valerolactone is converted to2-hydroxy-4-valerolactone.
 61. The method of claim 60, wherein the2-hydroxy-4-valerolactone is converted to angelica lactone.
 62. Themethod of claim 61, wherein the angelica lactone is reduced to4-valerolactone.
 63. The method of claim 61, wherein the angelicalactone is converted to methylene-methyl butyrolactone.
 64. The methodof claim 62, wherein the 4-valerolactone is converted tomethylene-methyl butyrolactone.